Therapeutic antibodies administered to a subject in need are often recognized as foreign by the subject's immune system. Even if the administered antibodies have been humanized, e.g. by grafting of murine CDRs into human immunoglobulin frameworks to minimize the mouse component, they still may elicit an immune response which compromises the efficacy and/or safety of the therapeutic.
According to the literature antibody responses in patients are dependent on the presence of both B-cell epitopes and T-cell epitopes. When a B-cell receptor recognizes and binds an antigen such as an administered therapeutic antibody, the antigen is internalized into the B cell by receptor-mediated endocytosis and undergoes proteolytic processing. The resulting peptides are subsequently presented by MHC class II molecules. Upon recognition of the T cell epitope by a T helper cell, the latter stimulates the corresponding B cells to proliferate and differentiate into antibody producing plasma cells.
In order to decrease the response of the patient's immune system to the administered antibodies, the prior art has provided several de-immunization techniques. Most of the current approaches focus on the removal of T-cell epitopes, whereas there are only limited examples of methods to reduce B-cell immunogenicity.
WO 93/18792 describes a process for the modification of antibodies by partial reduction of the antibody. This alters their immunogenicity so that their ability to induce an anti-isotypic response is selectively diminished, while they remain able to elicit an anti-idiotypic response. Albeit the method would be suitable for vaccines, anti-idiotypic responses are not desirable for other therapeutic applications.
Molineux G (2003) Pharmacotherapy 23: 35-85 describes the coupling of proteins to high-molecular-weight polyethylene glycol. However, Onda, M. et al (2008), PNAS Vol 105(32): 11311-11316 have reported a limited success of this approach with hybrid proteins composed of the variable fragment attached to a bacterial or plant toxin. Their hybrid proteins were inactivated; moreover, they found only a minor decrease in immunogenicity.
A second approach consists in chemotherapy prior to antibody administration, wherein patients are treated with cyclophosphamide or fludarabine. This approach is not desirable for the patients as the treatment damages the immune system (Kusher, B H et al (2007), Pediatr Blood Cancer 48: 430-434; Leonard J P et al (2005), J Clin Oncol 23: 5696-5704).
Nataga, S. and Pastan, I. (2009), Adv Drug Deliv Rev, p. 977-985 and Onda, M. et al (2008), PNAS Vol 105(32): 11311-11316 propose point mutations at “antigenic hot spots” on the foreign protein surface, thereby removing the B-cell epitope. They substituted bulky hydrophilic residues with large exposed areas by small amino acids (alanine, glycine and serine). Alanine is preferred for substitution as it is typically present in buried and exposed positions of all secondary structures and also does not impose new hydrogen bonding. Alanine lacks side chain atoms after the β-carbon that can react with antibodies and moreover maintains the conformation of the antigen. However, said “hot spots” described by Nataga and Pastan are conformational epitopes which are located in discrete clusters on the protein surface. Extensive experimental work is needed to determine the locations of the epitopes that could not be reproduced in a computer simulation and thus, their method does not represent a general solution to reduce immunogenicity of antibodies that can be applied routinely. Furthermore, a principle assumption of this method is that mainly hydrophilic residues on the molecular surface are involved in the contact with the host antibody. For most foreign proteins this is in fact true, however in cases were only portions (e.g. fragments, domains) of a naturally occurring protein is used, it may well be that also hydrophobic amino acids, formerly shielded by the contact to other domains become exposed to the solvent and present as epitope to the immune system. This is explicitly the case for Fv antibody fragments, where the interface residues on the variable domain are covered in the Fab fragment but are exposed in isolated variable domains. Currently available algorithms to predict B cell epitopes are poorly validated and typically have a low rate of success.
Thus, there is a need in the art to provide straight forward methods which effectively reduce the immunogenicity of antibody fragments and particularly for the variable domains.